Gene therapy for the thalassemia syndromes and sickle cell anemia requires that the introduced globin gene mimic the function of the in situ normal globin gene both in tissue specificity and efficiency of transcription. DNA-mediated gene transfer experiments have shown that efficient transcription of the human beta-globin transgene requires the presence of a far upstream enhancer. In the genome, this erythroid specific and development-stage independent enhancer is located in a DNase 1 super hypersensitive site (HS2) at -11Kb 5' of the embryonic epsilon globin gene and thus at -54 Kb 5' of the adult beta-globin gene. The HS2 enhancer directs the synthesis of erythroid specific, long enhancer transcripts which appear to play a pivotal role in enhancer function and transcriptional activation of the downstream cis-linked gene. The long-term objective of the proposed work is to study the molecular mechanism by which this far upstream enhancer activates across 54 Kb of DNA the embryonic, fetal and adult globin genes in erythroid cells at the respective developmental stage. The specific aim is to test the hypothesis that the synthesis of long enhancer transcripts by a tracking and transcription mechanism is required, and constitutes a precondition, for the transcriptional activation of the beta-like globin genes during erythropoiesis. The following proposed experiments are designed to test selected aspects of this hypothesis. (1) Assessment of the relative importance of the tracking v.s. the looping mechanisms in the function of the far upstream HS2 enhancer. The long enhancer transcripts, and thus the tracking mechanism, will be blocked by transcriptional terminators. The effect of this blockage on the expression of the cis-linked CAT gene will be studied. (2) Analysis of enhancer transcription in HS2-containing CAT plasmids transfected into erythroid cells at different developmental stages. (3) Detection of endogenous long enhancer transcripts in non-transfected K562 cells and primary human BFU- E's and CFU-E's. (4) Examination of the role in enhancer transcription of a 26 bp HS2 enhancer core and of the transcription factors that bind to the core. (5) Investigation of the synergistic effects of two neighboring DNase 1 hypersensitive sites HS3 and HS4 on the HS2-driven enhancer transcription. By a combination of oligonucleotide synthesis, PCR, recombinant CAT plasmids construction, transfection experiments, CAT assays and RNA protection assays, in the biological significance of the enhancer initiated upstream long transcript and thus of the tracking mechanism will be evaluated.